CN104480127A - Hyperthermophilic glycosidase mutant and application thereof in preparation of ginsenoside CK - Google Patents

Abstract

The invention relates to a hyperthermophilic glycosidase mutant and an application thereof in preparation of ginsenoside CK, and belongs to the technical field of biotechnology engineering. An amino acid sequence of the hyperthermophilic glycosidase mutant is as shown in SEQ ID NO:2; and the hyperthermophilic glycosidase mutant is applied to preparation of rare ginsenoside CK. The hyperthermophilic glycosidase mutant has high heat resistance and stability; relatively high catalytic activity can still be kept after heat preservation at 70-80 DEG C for over 300 hours, and thus hyperthermophilic glycosidase mutant has the advantages of low storage and transportation cost, and low requirement standards on a cooling system of a reactor when applied to production; the kinetic reaction is accelerated; meanwhile, the hyperthermophilic glycosidase mutant has beta-glucosaccharase activity; glucosides of main protopanaxadiol type saponins C3 site and C20 site can be hydrolyzed on the basis that the saponin structure is not destroyed; and the hyperthermophilic glycosidase mutant is efficient, specific, few in byproducts, high in yield and the like, so that the Fpglula can be applied to industrial production of a rare ginsenoside Compound K.

Description

Hyperthermophilic Glycosylase mutant and the application in Ginsenoside compound K preparation thereof

Technical field

The invention belongs to biotechnology field of engineering technology, be specifically related to one and derive from the genetic engineering bacterium that Fervidobacterium belongs to the hyperthermophilic Glycosylase mutator gene of (Fervidobacteium) bacterium, recombinant vectors, production mutant, and the application of this hyperthermophilic Glycosylase mutant in prepared by rare ginsenoside CK.

Background technology

Ginseng (Panax ginseng) is China's tradition rare traditional Chinese medicine, has a long history.The traditional medicine of China thinks that ginseng can " peace soul, only palpitation with fear, except perverse trend, the happy intelligence development of improving eyesight, clothes be made light of one's life by commiting suicide and prolonged life for a long time for main tonifying the five internal organs, peace spirit ".Ginsenoside is the main active substances in ginseng, and from ginseng, finds more than 100 kind of natural ginseng saponin(e product, is separated more than 40 and plants.The content of various ginsenoside is different, and various structures, pharmacologically active also has very large difference.Research finds that some rare ginsenoside that content is extremely low in ginseng (as Rg3, F2, Rh2, Compound K, Compound Mc etc.) just has very high pharmaceutical use and application prospect.

Rare ginsenoside CK (Compound K) be ginseng or ginsenoside oral after in mammalian and intraorganic main active component, in In vitro cell experiment and body, animal model experiment evidence shows, CK has anti-inflammatory, protect the liver, hypoglycemic and the important biomolecule such as anticancer active, Chinese food Drug Administration has ratified the clinical trial carrying out CK prevention and therapy arthritis.Thus rare ginsenoside CK has extremely important application potential in drug development etc.But do not contain or only contain the Ginsenoside compound K of trace in ginseng, limit its widespread use.Therefore based on saponin(e core skeleton and chemical structure similarity principle, the ginsenoside terminal saccharide that some content is high by being hydrolyzed, drug effect is low is prepared these rare ginsenosides in a large number and has just been become method the most feasible at present.The usual selectivity of chemical hydrolysis is poor, and productive rate is low, not easily purifies, and easily causes environmental pollution simultaneously.And Glycosylase hydrolysis rule possesses regioselectivity and the advantage such as stereoselectivity is high, yield is high, by product is few, pollution-free and easy suitability for industrialized production, be considered to prepare the potential method of rare ginsenoside most.

Derive from the thermophilic Glycosylase of thermophile bacteria, owing to having higher catalysis activity and thermostability, day by day receive the concern of people, become the focus of thermophilic Glycosylase fundamental research and development of new Glycosylase preparation.About Zimadzhunt L 340, after the first Zimadzhunt L 340 in 1985 and Taq archaeal dna polymerase are successfully used to polymerase chain reaction (PCR), the microorganism grown under special high thermal environment causes the attention of people just day by day.Many Zimadzhunt L 340s (thermophilic enzyme, 55-80 DEG C) with hydrolytic enzyme activities obtain exploitation in succession, and have played vital role in a lot of fields.In recent years, people are separated and obtain super Zimadzhunt L 340 (hyperthermophilicenzyme, 80-113 DEG C) from the Pyrococcus furiosus of oceanic heat flow, for modern enzyme engineering technology presents new application prospect.Zimadzhunt L 340 not only has the incomparable advantage of chemical catalyst, as high in catalytic efficiency and Substratspezifitaet by force, and the excellent stability of enzyme.Thus it can overcome middle temperature enzyme (mesophilic enzyme, 20-55 DEG C) and cold-adapted enzyme (psychrophilic enzyme,-2-20 DEG C) phenomenon of biological property instability that usually occurs in application process, thus a lot of high-temperature chemical reaction process is achieved, this will greatly promote the development of biotechnology industry, thus motivates technical transformation of the factory with financial strength the raising of level and quality of life.

Zimadzhunt L 340 is utilized to have the following advantages as biological catalyst: the preparation cost of (1) zymin reduces.Because the stability of Zimadzhunt L 340 is high, thus can at room temperature separating-purifying and packed and transported, and can keep active muchly.(2) kinetic reaction is accelerated.Along with the raising of temperature of reaction, molecular motion velocities is accelerated, and enzyme catalysis ability is strengthened.(3) the requirement standard of reactor cooling system is reduced, thus decrease energy consumption.(4) improve the purity of product.Under Zimadzhunt L 340 catalytic reaction condition (more than 70 DEG C), seldom there is miscellaneous bacteria to survive, thus decrease the pollution of bacterium metabolite to product.Because the pyroreaction of Zimadzhunt L 340 is active, and the stronger resistance to organic solvent, stain remover and denaturing agent, it is made all to be widely used in food, medicine, process hides, oil production and refuse process etc. potentiality.

Due to thermophile bacteria artificial culture difficulty, growth cycle is longer, and the content of Glycosylase is very low, is therefore unfavorable for directly utilizing thermophile bacteria to produce heat-resisting Glycosylase.

Summary of the invention

The invention provides a kind of hyperthermophilic Glycosylase mutant and the application in Ginsenoside compound K preparation thereof, to solve thermophile bacteria artificial culture difficulty, growth cycle is longer, and the content of Glycosylase is very low, is therefore unfavorable for directly utilizing thermophile bacteria to produce the problem of heat-resisting Glycosylase.

The technical scheme that the present invention takes is: hyperthermophilic Glycosylase mutator gene, and its nucleotide sequence is as described in SEQ ID NO:1.

The invention provides the recombinant plasmid and recombinant bacterial strain that build after being suddenlyd change by above-mentioned hyperthermophilic glycosidase genes.

The invention provides a kind of hyperthermophilic Glycosylase mutant Fpglu1a utilizing above-mentioned recombinant bacterial strain to prepare;

The aminoacid sequence of hyperthermophilic Glycosylase mutant of the present invention is as described in SEQ ID NO:2.

The invention provides the application of above-mentioned hyperthermophilic Glycosylase mutant in rare ginsenoside CK produces.

We are by the cultivation of wild-type thalline, full plasmid PCR, then obtain hyperthermophilic Glycosylase of the present invention sudden change recombinant plasmid, we entrust ancient cooking vessel state biology (Shanghai) to carry out gene sequencing, and sequencing result is as described in SEQ ID NO:1.

The hyperthermophilic glycosidase genes mutant plasmid obtained is imported in Escherichia coliBL21 (DE3) (purchased from Novagen company) host according to biology ordinary method, constructs mutant engineering bacteria.

With wild-type and mutant engineering bacteria for starting strain carries out microbial culture and IPTG induction, the wild-type needed for expression and mutant enzyme.Be intracellular enzyme through experimental verification target protein, thalline need be broken by the ultrasonication method of cell, then obtain highly purified target protein by methods such as high speed centrifugation, affinity chromatography purifying, SDS-PAGE polyacrylamide gel electrophoresises.

The Fervidobacterium being separated the natural extreme thermal environments such as self-heating fountain belongs to (Fervidobacterium) bacterium and belongs to thermophilic eubacte, belongs to bacterium be separated the Zimadzhunt L 340 obtained and have good zymologic property and application potential more from Fervidobacterium.To more piece Fervidobacterium (Fervidobacterium pennivorans DSM9078) genomic analysis, we find to comprise several thermophilic glycosidase genes in the genome of this bacterium.Applied molecular biology technology, we have carried out cloning and expressing to one of them glycosidase genes mutant, expressing protein has activity of beta-glucosidase higher compared with wild-type, further enzymology confirms, this albumen is a kind of hyperthermophilic beta-glucosidase of good properties.

We have carried out vitality test experiment to the wild-type hyperthermophilic Glycosylase of colibacillus engineering expression and mutant thereof, confirm that they have very high hydrolytic activity to p-nitrophenyl-β-D-Glucose glycosides and p-nitrophenyl-β-D-galactoside.And mutant hydrolysis is 2.5 times of wild-type to the hydrolysis vigor of p-nitrophenyl-β-D-Glucose glycosides.Compared with some other Glycosylase, hyperthermophilic Glycosylase mutant has very strong thermostability, and when temperature reaches 100 DEG C, vigor is lasting rises, and at 70 DEG C and 80 DEG C, insulation still can keep the vigor of more than 80% more than 300 hours, did not reach the transformation period; Enzyme liquid can reach 3.5 hours the transformation period of 90 DEG C.Thus Fpglu1a belongs to hyperthermophilic enzyme, there is zymin cost in the application low, there is carrying cost low, accelerate kinetic reaction, low to reactor cooling systematic requirement criteria, there is activity of beta-glucosidase simultaneously, and can on the basis not destroying saponin(e structure, the glucoside of hydrolysis main diol type ginsenoside C3 position and C20 position generates rare ginsenoside Compound K, have efficient, single-minded, by product is few, productive rate advantages of higher, thus Fpglu1a can be applied in the industrial production of rare ginsenoside Compound K, for its industrial applications is laid a good foundation.

By transforming the research of rare ginsenoside to hyperthermophilic Glycosylase mutant, this is thermophilic, and Glycosylase can be hydrolyzed diol type ginsenoside Rb1, Rb2 and Rc, converted product utilizes HPLC and LC-MS to carry out identifying quickly and accurately, result shows, Fpglu1a transforms ginsenoside Rb1, and the end product of Rb2 and Rc is Ginsenoside compound K.

Accompanying drawing explanation

Fig. 1 is hyperthermophilic Glycosylase wild type gene PCR primer agarose gel electrophoresis figure;

Left road: DNA molecular Marker DL5000; Right road: the pcr amplification product of hyperthermophilic Glycosylase wild type gene, there is obvious bright band in right road at molecular weight 1000bp place, with expection molecular weight 1398bp conform to, so we by PCR method fish the gene got to be hyperthermophilic Glycosylase wild type gene described in this patent.

Fig. 2 is plasmid regrouping process figure;

Fig. 3 is hyperthermophilic Glycosylase and mutant denaturing electrophoretic analysis chart thereof, from left to right successively: protein molecular weight Marker, and the wild-type enzyme of the thick enzyme of ultrasonication and affinity chromatography gained and mutant enzyme;

Fig. 4 is that temperature is to hyperthermophilic Glycosylase mutant effect of vigor graphic representation;

Fig. 5 is that pH is to hyperthermophilic Glycosylase mutant effect of vigor graphic representation.

Fig. 6 is the HPLC figure that hyperthermophilic Glycosylase mutant transforms ginsenoside Rb1's different time; The wherein HPLC figure of (A) eight kinds of ginsenoside standard substance, (B) mutant transform ginsenoside Rb1 0 hour HPLC figure, (C) mutant transform ginsenoside Rb1 0.5 hour HPLC figure, (D) mutant transform ginsenoside Rb1 12 hours HPLC figure;

Fig. 7 is the HPLC figure that hyperthermophilic Glycosylase mutant transforms Ginsenoside Rb2's different time; The wherein HPLC figure of (A) eight kinds of ginsenoside standard substance, (B) mutant transform Ginsenoside Rb2 0 hour HPLC figure, (C) mutant transform Ginsenoside Rb2 6 hours HPLC figure, (D) mutant transform ginsenoside Rb1 36 hours HPLC figure;

Fig. 8: hyperthermophilic Glycosylase mutant transforms the HPLC figure of Ginsenoside Rc's different time; The wherein HPLC figure of (A) eight kinds of ginsenoside standard substance, (B) mutant transform Ginsenoside Rc 0 hour HPLC figure, (C) mutant transform Ginsenoside Rc 8 hours HPLC figure, (D) mutant transform Ginsenoside Rc 36 hours HPLC figure;

Fig. 9 is the electron spray(ES) ion massspectrum figure of Rc converted product 1;

Figure 10 is the mass spectrum series connection bar graph of Rc converted product 1;

Embodiment

The structure of embodiment 1 thermophilic bacterium glycosidase engineering and the expression of enzyme thereof

(1) cultivation of thermophilic bacterium Fervidobacterium pennivorans DSM 9078 and the extraction of chromosomal DNA thereof

Thermophilic bacterium Fervidobacterium pennivorans DSM 9078 is the culture medium prescription provided according to DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbHGerman Collection of Microorganisms and Cell Cultures) purchased from DSMZ (Germany) culture condition, and the substratum moiety of often liter of Fervidobacterium pennivorans DSM 9078 is as follows: NH ₄c1,0.5g; MgSO ₄× 7H ₂o, 0.16g; KH ₂pO ₄, 1.6g; Na ₂hPO ₄× H ₂o, 1.0g; CaCl ₂× 2H ₂o, 0.06g; Tracemineral solution, 10ml; Vitamin solution, 10ml; Yeast extract, 2g; Trypticase, 2g; Resazurin, 0.5mg; Glucose, 3g; Cysteine-HCl × H ₂o, 0.3g; Na ₂s × 9H ₂o, 0.3g.Wherein often liter of tracemineral solution's is composed as follows: nitrilotriacetic acid, 1.5g; MgSO ₄× 7H ₂o, 3g; MnSO ₄h ₂o, 0.5g; NaC1,1.0g; FeSO ₄7H ₂o, 0.1g; CoSO47H ₂o, 0.18g; CaC1 ₂2H ₂o, 0.1g; ZnSO ₄× 7H ₂o, 0.18g; CuSO ₄× 5H ₂o, 0.01g; KAl (SO ₄) ₂× 12H ₂o, 0.02g; H ₃bO ₃, 0.01g; Na ₂moO ₄2H ₂o, 0.01g; NiCl ₂× 6H ₂o, 0.03g; Na ₂seO ₃× 5H ₂o, 0.3g.The formula of often liter of Vitamin solution is as follows: Biotin, 2mg; Folic acid, 2mg; Pyridoxine-HCl, 10mg; Thiamine-HCl × 2H ₂o, 5mg; Riboflavin, 5mg; Nicotinic acid, 5mg; D-Ca-pantothenate, 5mg; Vitamin B12,0.1mg; P-Aminobenzoic acid, 5mg; Lipoic acid, 5mg.The stem cell of Fervidobacterium pennivorans DSM 9078 (culture presevation DSM9078) adds the above-mentioned substratum of 1ml, is transferred in the culture tube that the fresh above-mentioned substratum of 10mL is housed after resuspended, and rotating and culturing pipe is with mixing a little; Pass into nitrogen 3 minutes to eliminate the oxygen in culture tube, to be placed in high-temperature cultivation case 65 DEG C of static gas wave refrigerator 2 days.Then to be transferred in the Anaerobic culturel bottle that the above-mentioned substratum of 100ml is housed 65 DEG C of static gas wave refrigerator 2 days, 8000rpm collects above-mentioned thermophilic thalline in centrifugal 20 minutes, and-40 DEG C to preserve thalline for subsequent use

Get the thalline of above-mentioned collection, application bacterial genomes DNA extraction kit (purchased from the clean biotech firm of Hangzhou Wei Te) extracts bacterial genomes DNA to specifications, by its purity of DNA gel electrophoresis detection and the concentration of 0.8%, and save backup in-20 DEG C.

(2) Cloning and Expression of the wild-type alpha-glucosidase gene of Fervidobacterium pennivorans DSM 9078 is derived from.

Primer is designed according to sequence (sequence number X74163),

Primer 1:5'-CAGCAG cATATGtTTCCAAAGTCATTTATG-3'(underscore place is the restriction enzyme site of NdeI);

Primer 2: 5'-CGAGCC cTCGAGtTAGAATTTCATCAAATTG-3'(underscore place is the restriction enzyme site of XhoI).

Restriction enzyme site set by two primers and NdeI and XhoI of expression vector pET28a match, and are suitable at E. coli.

PCR reacts: containing 0.5 μ l Ex-Taq archaeal dna polymerase in 50 μ l reaction systems, 5 μ l Ex-Taq DNA polymerase buffer liquid, 1 μ l genomic dna, 1.5 μ l dNTP mixtures (often kind of nucleotide concentration 25nmol/L), 1 μ l upstream primer, 1 μ l downstream primer, the aseptic ultrapure water of 40 μ l.Often 94 DEG C of sex change 1 minute in circulation, 61.8 DEG C of annealing 1 minute, 72 DEG C extend 1 minute, and last circulation is extended down to 10min, totally 30 circulations.Detect PCR primer with 1.0% agarose gel electrophoresis, molecular weight is consistent with (1398) of expection, as shown in Figure 1.

Use PCR primer purification kit (the PCR Clean-up Kit that Bay Gene company produces, step is as follows: in PCR solution, add the Buffer PCR-A that triploid is long-pending, mixing; Above-mentioned solution is joined in DNA-prep pillar, centrifugal 30 seconds of 12000rpm; In DNA-prep pillar, add 500 μ lBuffer W2, centrifugal 1 minute of 12000rpm, then repeat once; DNA-prep posts transfer to clean 1.5ml microfuge tube, add the deionized water of 30 μ l sterilizings, the centrifugal PCR primer namely obtaining purifying for 1 minute of 12000rpm after room temperature places one minute) purifying is carried out to amplified production ,-20 DEG C save backup.

The PCR primer restriction enzyme NdeI enzyme of purifying is cut (the above-mentioned PCR primer of 20ul reaction system: 10ul, 5ul deionized water, 2ul NEBuffer 4 damping fluid, 1ul NdeI enzyme), after being incubated 1 hour at 37 DEG C, directly add 1 μ l XhoI, then be incubated 1 hour at 37 DEG C.Enzyme cuts complete, electrophoresis on the sepharose of 1.0%, application DNA gel detection kit reclaim enzyme cut after DNA fragmentation.The DNA fragmentation size that the agarose nucleic acid gel electrophoresis detection gel of 1.0% reclaims is about 1398bp, conforms to, DNA for the purpose of gained DNA is described with target DNA fragment 1398bp size.

By pET-28a carrier (coli expression carrier, containing T7 strong promoter, C/N-terminal Histidin Tag and kalamycin resistance gene etc.) cut (20ul reaction system: 10ul pET-28a carrier with restriction enzyme NdeI enzyme, 5ul deionized water, 2ul NEBuffer 4 damping fluid, 1ul NdeI enzyme), after being incubated 1 hour at 37 DEG C, directly add 1 μ l XhoI, then be incubated 1 hour at 37 DEG C.Enzyme cuts complete, then uses alkaline dephosphorylation enzyme (CIAP) to process, in 0.8% sepharose, detect linear carrier and enzyme purification cut after carrier.Connect at 16 DEG C of application T4DNA ligase enzymes the glycosidase genes fragment and carrier segments that enzyme cuts, obtain recombinant vectors (building process is as Fig. 2).

(3) Cloning and Expression of the glucoside enzyme mutant gene of Fervidobacterium pennivorans DSM 9078 is derived from.Design mutant primer, specifically

Primer 1:5'-GATACTCCT GCG GAATTTGCAAAATAC-3';

Primer 2: 5'-TGCAAATTC CGC AGGAGTATCATCAC-3'.

Full plasmid PCR reaction: containing 1 μ l Ex-Taq archaeal dna polymerase in 50 μ l reaction systems, 5 μ l Ex-Taq DNA polymerase buffer liquid, 1 μ l genomic dna, 4 μ l dNTP mixtures (often kind of nucleotide concentration 25nmol/L), 1 μ l upstream primer, 1 μ l downstream primer, the aseptic ultrapure water of 37 μ l.Often 95 DEG C of sex change 40 seconds in circulation, 62 DEG C of annealing 1 minute, 68 DEG C extend 8 minutes, and last circulation is extended down to 10min, totally 30 circulations.Detect PCR primer with 0.8% agarose gel electrophoresis, molecular weight is consistent with (6000bp) of expection.

Use PCR primer purification kit (the PCR Clean-up Kit that Bay Gene company produces) to carry out purifying to amplified production ,-20 DEG C save backup.

The PCR primer Dpn1 enzyme of purifying is carried out digest (the above-mentioned PCR primer of 50ul reaction system: 30ul, 14ul deionized water, 5ul 10 × T Buffer 4 damping fluid, 1ul Dpn1 enzyme), for transforming after being incubated 1 hour at 37 DEG C.

(4) Expression and purification of wild-type and mutant

Recombinant plasmid is transferred in the competent cell of bacillus coli DH 5 alpha, carries out dull and stereotyped preliminary screening.Picking list bacterium colony, cultivates in 5ml LB substratum.After utilizing PCR to identify positive colony, gene sequencing is carried out to goal gene, and proves accurate.In order to express target protein, the plasmid of successful connection being transferred in expression strain E.coli BL21 (DE3) CodonPlus competent cell and expressing.LB substratum is composed as follows: Bacto-Tryptone, and 1.0%; Bacto-Yeast Extract, 0.5%; NaCl, 0.5%.

Recombinant bacterium is inoculated in 5ml and receives in the LB substratum of mycin by the inoculum size of 2% containing 100 μ g/ml cards, 37 DEG C, shakes overnight incubation.Connect 200ml LB substratum by same inoculum size, 37 DEG C of concussions are cultivated until OD ₆₀₀when reaching about 1.0, add the inductor IPTG that final concentration is 1mM, overnight induction at 30 DEG C, after centrifugal 20 minutes of 8000rpm, collecting and obtain thalline, preserving in-20 DEG C of refrigerators, for extracting target protein.

This patent uses Sodium phosphate dibasic-citric acid (pH7.0) damping fluid as the resuspended damping fluid preparing crude enzyme liquid.Thalline (5g) adds 30ml 20mM Sodium phosphate dibasic-citric acid (pH7.0) damping fluid by 1:6 (w/v), after ultrasonication about 1 hour (3s × 3s), ie in solution becomes limpid, centrifugal 20 minutes of 10000rpm, collects the crude enzyme liquid that supernatant obtains recombinase.

Crude enzyme liquid uses the method for nickel affinity chromatography to carry out abstraction and purification to restructuring Glycosylase, the pure Zimadzhunt L 340 obtained is combined with chromatography column with the imidazole solution wash-out of 150mM, the purity of application SDS-PAGE (10%) electrophoresis detection recombinant protein, result as shown in Figure 1, be followed successively by protein molecular weight Marker from left to right, the wild-type enzyme of the thick enzyme of ultrasonication and affinity chromatography gained and mutant enzyme, all there is a single electrophoretic band in wild-type and mutant zymoprotein as can be seen here near 33000Da.

The characteristic of embodiment 2 hyperthermophilic restructuring Glycosylase mutant

(1) substrate specificity of wild-type and mutant Glycosylase

Take various p-NP glycoside substrates, carry out enzyme activity determination with reference to (1) described Glycosylase condition determination.

As shown in Table 1, thermophilic Glycosylase and mutant thereof show different hydrolysis vigor to serial p-NP glycoside substrates, the vigor being wherein hydrolyzed p-nitrophenyl-β-D-Glucose glycosides is the highest, and the vigor of mutant hydrolysis p-nitrophenyl-β-D-Glucose glycosides is about 2.5 times of wild-type.

Table 1 wild-type hyperthermophilic Glycosylase and mutant substrate selective thereof

(2) transformation efficiency that the substrate specificity catalysis ginsenoside of wild-type and hyperthermophilic Glycosylase mutant Glycosylase transforms compares

Application HPLC analyzes the transformation efficiency investigating wild-type and mutant Glycosylase conversion ginsenoside substrate respectively, and result is as table 2, and compared with wild-type, mutant glycosidases catalyze ginsenoside Rb1, the transformation efficiency of Rb2 and Rc improves 38%, 42% and 20% respectively.As can be seen here, transgenation is to the raising Be very effective of this Glycosylase vigor, and the transformation efficiency transforming ginsenoside improves obviously.

Table 2: the transformation efficiency that wild-type hyperthermophilic Glycosylase and mutant catalysis ginsenoside thereof transform compares

(3) optimal reactive temperature

Temperature of reaction is the important factor affecting enzyme catalysis vigor.Generally speaking, thermophilic Glycosylase reaction vigor is at high temperature far away higher than low temperature, but the stability near optimum temperuture but reduces greatly, this patent has investigated the effect of vigor of temperature to thermophilic Glycosylase mutant in 30-100 DEG C of temperature range, p-nitrophenyl-β-D-Glucose glycosides (p-nitrophenyl-β-d-glucopyrinoside) solution that use final concentration is 0.2mM is as substrate, with the relative activity that enzyme is lived, temperature is mapped, as seen from Figure 4, mutant vigor 100 DEG C time still continues to raise, and thus this enzyme belongs to hyperthermophilic enzyme.

The detection of enzymic activity: reaction system is 1ml, buffer system is 20mM Sodium phosphate dibasic-citrate buffer solution (pH7.0), adds the enzyme of 5ul 0.1mg/ml, and 80 DEG C measure OD in 1 minute ₄₀₅increased value, obtain the slope of straight line, be the vigor of enzyme.1 enzyme activity unit is defined as 1 minute hydrolysis substrate and generates 1 μm of ol p-NP (ε=0.016 μM ^-1.cm ^-1) required for enzyme amount.

(4) optimal pH

Environmental pH can affect the dissociated state of Charged acids and the conformation of enzyme in enzyme molecule, and then affects the catalysis activity of enzyme.PH measurement range is between 3.0 ~ 10.6, three kinds of buffer systems are selected to be Sodium phosphate dibasic-citrate buffer solution (pH 3.0-8.0) respectively, Tris-HCl damping fluid (pH7.5-9.0) and glycine-NaOH (pH 8.5-10.5).With the relative activity that enzyme is lived, pH is mapped, as seen from Figure 5, thermophilic Glycosylase Fpglu1a has good catalysis activity in neutral conditions, its optimal pH is 7.0, the vigor of 50% still can be reached under meta-alkalescence condition, but catalysis activity is lower under strongly acidic conditions, above analysis shows that this Zimadzhunt L 340 is neutral thermophilic Glycosylase.

(5) temperature stability

Be that the pure enzyme liquid of 1.0mg/ml is placed in damping fluid (20mMpH7.0 Sodium phosphate dibasic-citrate buffer solution) insulation under differing temps (70 DEG C, 80 DEG C and 90 DEG C) by concentration, measure the remaining vigor of different soaking time.This enzyme is incubated the vigor that still to keep more than 80% more than 300 hours at 70 DEG C and 80 DEG C, does not reach the transformation period; And 3.5 hours can be reached 90 DEG C of transformation period.More than analyze and show that thermophilic Glycosylase Fpglu1a has extraordinary thermostability.

Embodiment 3 is thermophilic, and Glycosylase Fpglu1a transforms rare ginsenoside CK

By the solution of 1mL 1mg/ml ginsenoside Rb1, Rb2 and Rc, add isopyknic purifying enzyme liquid obtained above again, react 12 hours under 70 DEG C of conditions, in reaction system, isopyknic water-saturated n-butanol termination reaction is added after reaction, the centrifugal 2min of 8000rpm after vortex mixing, upper strata n-butanol layer solution is got after leaving standstill 5min, 60 DEG C of water bath methods, collect solid sample, use chromatogram rank methanol constant volume to 1ml, obtain sample after Radix Ginseng total saponins's bio-transformation, detect for HPLC with after organic membrane filtration of 0.22 μm.High performance liquid chromatography testing conditions is, C ₁₈reverse-phase chromatographic column (5cm × 3.0mm, 2.7 μm; Supelco.USA).Moving phase is water (A) and acetonitrile (B), condition of gradient elution: 0min, 85 (A): 15 (B); 5-10min, 81:19; 10-13min, 81:19; 13-16min, 75:25; 16-20min, 64:36; 20-25min, 55:45; 25-28min, 35:65; 28-35min, 20:80; 35-40min, 100% (B).Flow velocity 1.0ml/min.Determined wavelength: 203nm.Column oven: 35 DEG C.Sample size: 5 μ l.Standard substance ginsenoside is provided by Nanjing Zelang Pharmaceutical Technology Inc..Application+ESI ionogenic Agilent 6520Q-TOF mass spectrum determines the molecular weight of converted product further.Mass Spectrometry Conditions: dry gas temperature 350 DEG C, dry gas flow velocity 4l/min, atomization gas pressure 30psig, capillary voltage 3500V, transmission voltage 350V, taper hole voltage 65V.One-level spectrogram acquisition rate is respectively 2spectra/s.Collision gas is N2.Before sample test, use tuning liquid correction mass axle.

By contrasting with standard substance retention time, we can determine that the converted product of ginsenoside Rb1 and Rb2 is Ginsenoside Rd and CK, and Ginsenoside Rd is finally converted into CK (Fig. 6, A-D; Fig. 7, A-D); The converted product of Ginsenoside Rc, after HPLC and LC/MS qualification (Fig. 9,10), be respectively ginsenoside CMc, Rd and CK, and ginsenoside CMc and Rd is finally converted into CK (Fig. 8, A-D).To sum up, mutant can by ginsenoside Rb1, Rb2 and Rc is converted into Ginsenoside compound K.Ginsenoside Rb1, Rb2 and Rc is the main component in ginseng water-soluble crude extract, and the three kinds of main saponin(es of this in crude extract can be finally converted into rare ginsenoside CK by mutant, and utilizes single saponin(e for compared with conversion of substrate, greatly reduces conversion cost.And the high stability of mutant and high efficiency make it have great advantage in industrial application.

The extraction and isolation of embodiment 4 converted product

Detect through HPLC, in the reactant for reforming alcohol extract of 10L, contain purity is the Ginsenoside compound K of 60.9%, we utilize HP20 resinification to carry out just being separated to alcohol extract, Ginsenoside compound K purity after separation can reach 80%, after HP20 takes off hydration, primary extract carries out essence through H41 resinification and carries, and final acquisition purity reaches the Ginsenoside compound K of 92.6%.

In above technical scheme, all basic biologic operations are all with reference to " Molecular Cloning: A Laboratory guide " (third edition, Science Press, 2002).

The hyperthermophilic Glycosylase mutant catalyzed reaction substrate spectrum used used in the present invention is not limited to glucosides class, in preferred embodiments, the present invention uses p-nitrophenyl-β-D-Glucose glycosides (p-nitrophenyl-β-d-glucopyrinoside) as substrate.

Content disclosed in this invention, believes that those skilled in the art can apply the present invention to greatest extent.Therefore the preferred specific embodiments before should be understood to only illustrate, but not limits the scope of the invention by any way.This area researchist, when not departing from its purport and scope, can carry out various change and improvement to the present invention.

Claims (7)

1. a hyperthermophilic Glycosylase mutator gene, its nucleotide sequence is as described in SEQ ID NO:1.

2. the recombinant expression vector be made up of hyperthermophilic Glycosylase mutator gene according to claim 1, is characterized in that: recombinate with expression vector pET-11a, pET-28a or pET-20b.

3. the recombinant expression vector of hyperthermophilic Glycosylase mutator gene formation as claimed in claim 2, is characterized in that: expression vector is pET-28a.

4. one kind imports the recombinant bacterial strain obtained by the recombinant expression vector described in Claims 2 or 3, it is characterized in that: this recombinant expression vector is imported in Escherichia coli BL21 (DE3) CodonPlus host cell or Escherichia coli BL21 (DE3) host cell, obtain recombinant bacterial strain.

5. recombinant bacterial strain as claimed in claim 4, is characterized in that: host cell is Escherichia coliBL21 (DE3).

6. a hyperthermophilic Glycosylase mutant, its aminoacid sequence is as described in SEQ ID NO:2.

7. hyperthermophilic Glycosylase mutant according to claim 6 is preparing the application in rare ginsenoside CK.